Kinetic deuterium isotope effects for 7-alkoxycoumarin O-dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes Rate-limiting C-H bond breaking in cytochrome P450 1A2 substrate oxidation Keon-Hee Kim 1 , Emre M. Isin 2 , Chul-Ho Yun 1 , Dong-Hyun Kim 3 and F. P. Guengerich 2 1 Hormone Research Center and School of Biological Sciences and Technology, Chonnam National University, Gwangju, Korea 2 Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN, USA 3 Doping Control Center, Korean Institute of Science and Technology, Seoul, Korea Cytochrome P450 (P450, EC 1.14.14.1) enzymes catalyze the oxidation of a great variety of steroids, fat-soluble vitamins, eicosanoids, and numerous xeno- biotic chemicals including drugs, natural products, carcinogens, pesticides, and other compounds [4,5]. The study of these enzymes has been facilitated by the availability of artificial substrates that can be utilized as probes because of their spectral and fluorescent properties. Among these are the coumarins [6,7] and resorufins [7,8]. Coumarin and several derivatives are Keywords alkoxycoumarins; coumarins; Cytochrome P450; kinetic isotope effects; microsomal reactions Correspondence F. P. Guengerich, Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232–0146, USA Fax: +615 322 3141 Tel.: +615 322 2261 E-mail: f.guengerich@vanderbilt.edu Note The conventions used for kinetic hydrogen isotope effects are D k ¼ intrinsic kinetic deuterium isotope effect, D V ¼ H k cat ⁄ D k cat , and D (V ⁄ K) ¼ ( H k cat ⁄ H K m ) ⁄ ( D k cat ⁄ D K m ) [2,3]. (Received 20 February 2006, accepted 17 March 2006) doi:10.1111/j.1742-4658.2006.05235.x 7-Ethoxy (OEt) coumarin has been used as a model substrate in many cyto- chrome P450 (P450) studies, including the use of kinetic isotope effects to probe facets of P450 kinetics. P450s 1A2 and 2E1 are known to be the major catalysts of 7-OEt coumarin O-deethylation in human liver micro- somes. Human P450 1A2 also catalyzed 3-hydroxylation of 7-methoxy (OMe) coumarin at appreciable rates but P450 2E1 did not. Intramolecular kinetic isotope effects were used as estimates of the intrinsic kinetic deuter- ium isotope effects for both 7-OMe and 7-OEt coumarin dealkylation reac- tions. The apparent intrinsic isotope effect for P450 1A2 (9.4 for O-demethylation, 6.1 for O-deethylation) showed little attenuation in other competitive and noncompetitive experiments. With P450 2E1, the intrinsic isotope effect (9.6 for O-demethylation, 6.1 for O-deethylation) was attenu- ated in the noncompetitive intermolecular experiments. High noncompeti- tive intermolecular kinetic isotope effects were seen for 7-OEt coumarin O-deethylation in a baculovirus-based microsomal system and five samples of human liver microsomes (7.3–8.1 for O-deethylation), consistent with the view that P450 1A2 is the most efficient P450 catalyzing this reaction in human liver microsomes and indicating that the C-H bond-breaking step makes a major contribution to the rate of this P450 (1A2) reaction. Thus, the rate-limiting step appears to be the chemistry of the breaking of this bond by the activated iron-oxygen complex, as opposed to steps involved in the generation of the reactive complex. The conclusion about the rate-limit- ing step applies to all of the systems studied with this model P450 1A2 reac- tion including human liver microsomes, the most physiologically relevant. Abbreviations b 5 , cytochrome b 5 (b 5 , 1.6.2.2); di-12 : 0 GPC, L-a-dilauroyl-sn-glycero-3-phosphocholine; MS, mass spectrometry; OEt, ethoxy; OMe, methoxy; OR, alkoxy; P450, cytochrome P450 (also termed ‘heme-thiolate protein P450’ [1]). FEBS Journal 273 (2006) 2223–2231 ª 2006 The Authors Journal compilation ª 2006 FEBS 2223 natural products themselves, but the artificial 7-alkoxy derivatives have been particularly useful because of the large fluorescent changes that occur upon hydroxyla- tion of the alkyl group and release to form 7-hydroxy- coumarin (umbelliferone) [6,7]. Kinetic hydrogen isotope effects, especially with deu- terium, have been used to probe aspects of the cata- lytic mechanisms of many enzymes, including P450s [9–12]. A basic concept is that the existence of a non- competitive intermolecular kinetic deuterium isotope effect argues that the C-H bond-breaking step is at least partially rate-limiting [2,3,13]. If an alternate reaction path is possible, then the impedance of one pathway by deuterium substitution may yield a ‘meta- bolic switch’ (or ‘isopically sensitive branching’) [14] to produce enhanced levels of the other product, although this is not always the case due to kinetic issues. Both high and low kinetic deuterium isotope effects have been reported for various P450 reactions. Although early work in the field suggested that high isotope effects were uncommon with P450s, particularly in microsomes [9,15,16], in more recent work a case may be made that a significant contribution of the C-H bond-breaking step to overall rates of catalysis may be more the norm than the exception, at least in purified enzyme systems [17]. For instance, kinetic deuterium isotope effects as high as 15 have been reported with rabbit P450 1A2 [18]. Human P450 2A6 showed high intramolecular kin- etic isotope effects for the O-dealkylation of 7-OMe and 7-OEt coumarin (10 and 6, respectively) (Fig. 1), which were not attenuated in noncompetitive experi- ments, arguing that C-H bond breaking is a rate-limit- ing step [19]. However, P450 2A6 does not make a substantial contribution to this particular enzyme activity in liver microsomes, and the question can be raised as to whether the microsomal (and in vivo) rates are limited by reduction and other steps involved in the generation of the reactive oxygen species, as opposed to the reactions of the activated complex with substrates, in light of the low endogenous concentra- tion of NADPH-P450 reductase [20]. In earlier work, the laboratory of Lu and Miwa [10,21,22] reported high intrinsic kinetic deuterium isotope effects for 7-OEt coumarin O-deethylation by some rat P450 enzymes (today known as P450s 2B1 and 1A1). How- ever, these high isotope effects were strongly attenu- ated in either intermolecular noncompetitive studies with the purified enzymes or with liver microsomes, except in the case of liver microsomes prepared from 3-methylcholanthrene-treated hamsters (in this case the enzyme was not purified) [22]. We analyzed two human liver P450s implicated in 7-OEt coumarin O-deethylation, namely P450s 1A2 and 2E1 [23]. 7-OMe coumarin was also used as a deuterated probe, to avoid the issue of prochirality inherent in the ethyl group. We found high isotope effects expressed even in the noncompetitive experi- ments performed in microsomes. A major fraction of an alternate product, derived from 3-hydroxylation, was found with these enzymes and in microsomes. The results are interpreted in the context of a rate-limiting chemical step involved in the reaction of the activated enzyme complex (Fe-O species) with the substrate, even in the most relevant biological system, human liver microsomes. Results and discussion Intramolecular kinetic isotope effects The intramolecular kinetic isotope effects measure the comparative rates for cleavage of a C-H bond and a C-D bond at a single carbon [3]. The value obtained from such an experiment can be used as an approxi- mation of the true intrinsic kinetic deuterium isotope effect and for comparison with all other types of experiments to ascertain the extent to which any attenuation has occurred [3]. The intermolecular experiments, the studies which provide the most infor- mation about the rate-limiting steps (see below), must be considered in the context of the estimated intrinisic isotope effects. The values for 7-OMe O-demethylation, measured by MS, were 9.4 and 9.6 for P450s 1A2 and 2E1, respectively (Table 1). The corresponding values were 6.1 for both P450s 1A2 and 2E1 with 7-OEt coumarin (Table 1). Whether these values should necessarily be identical to each other (for the two enzymes) is not clear. In one sense they should be independent of what the rate-limiting steps are for these two enzymes (and P450 2A6 [19]), if similar mechanisms are operative. At least two factors can perturb these values so that they are not true intrinsic kinetic deuterium isotope effects. One is the issue of prochirality in the case of 7-OEt coumarin. That is, the two methylene hydrogens are not equivalent, and we are using a racemic Fig. 1. Oxidation reactions with coumarins catalyzed by P450s. P450 coumarin dealkylation isotope effects K H. Kim et al. 2224 FEBS Journal 273 (2006) 2223–2231 ª 2006 The Authors Journal compilation ª 2006 FEBS mixture. Conceivably the P450s being examined here could show a partial or strict stereochemical preference for one or the other face. For instance, partial selectiv- ity has been seen in oxidations of ethylbenzene [24] and a substituted nitrosamine [25]. If strict stereoselec- tivity of R versus S hydrogens occurs with 7-OEt coumarin, then the experiment effectively becomes an intermolecular competitive experiment (see below). Studies with 7-OMe coumarin have an advantage in that there is no issue with pro-chirality. However, with both d 1 7-OEt coumarin and d 2 7-OMe coumarin some perturbation can occur because of a geminal secondary kinetic isotope effect. These values are traditionally £ 1.2 for each deuterium atom [26], and only limited literature is available about P450 (gem) secondary kinetic isotope effects [19,27–29]. The existence of a gem secondary kinetic isotope effect has the effect of attenuating the rate of C-H bond-breaking, so in prin- ciple the intrinsic kinetic isotope effect would be even higher than that estimated by this method (assuming a secondary isotope effect > 1). With d 2 7-OMe couma- rin, the secondary effects would be multiplicative [26], and if a secondary isotope effect as high as 1.2 existed, it would rise to (1.2) 2 ¼ 1.44. Although we cannot readily estimate the secondary isotope effect, the values of the intramolecular values in Table 1 are reasonable but should be considered lower estimates of the intrin- sic kinetic deuterium isotope effects. Intermolecular competitive kinetic isotope effects The intermolecular competitive kinetic isotope effects were estimated using MS of the products (Table 1). With 7-OEt coumarin, the value was high for P450 1A2 (9.5); with 7-OMe coumarin the value for P450 1A2 was 10.6 and for P450 2E1 was 7.7. The value of these measurements lies in their comparison with the values measured in the intramolecular experi- ments, i.e. the estimates of the intrinsic kinetic isotope effects (Table 1, see above). The value for P450 1A2 was not diminished, within experimental error and considering the caveats about secondary isotope effects mentioned above, but some attenuation was seen for P450 2E1, from 7.7 (± 0.5) to 4.2 (± 1.0) (Table 1). Attenuation of an intrinsic isotope effect (estimated with the intramolecular experiment in Table 1) in a competitive experiment provides evidence that the rate of exchange of substrates is a slow process relative to forward progress past an irreversible step. That is, if an isotope effect is sensed in the enzyme, the enzyme will process the deuterated substrate if exchanging that (deuterated) substrate for a protiated one takes longer (than the C-H bond cleavage process). Non-competitive intermolecular isotope effects The noncompetitive intermolecular isotope effects were measured by running assays with d 0 and perdeuterated (at the carbon being oxidized) substrates and compar- ing the v versus S plots (Figs 2 and 3, and Tables 2 and 3). The patterns were very similar for 7-OMe and 7-OEt coumarin, with P450 1A2 showing high isotope effects (8–16) and P450 2E1 yielding isotope effects of 2–4, allowing for the variability in both cases. For P450 2E1, the discernment of k cat and K m components was difficult (Fig. 3). With P450 1A2, 3-hydroxylation was observed (Tables 2 and 3), with the catalytic efficiency being comparable to that of O-dealkylation (see Supplement- ary material). These products were also observed in reactions with P450 2A6 [19] although with less effi- ciency than O-dealkylation. (3-Hydroxylation has also been reported in human [30] and other [31] liver micro- somes, but rates were not reported.) As shown in Figs 2 and 3 and Tables 2 and 3, deu- teration of the alkoxy group had little tendency to divert the reaction to 3-hydroxylation (Fig. 1), even Table 1. Kinetic isotope effects for 7-OR coumarin O-dealkylation by purified P450s estimated by MS. Kinetic isotope effect Intermolecular competitive Intramolecular (noncompetitive) P450 7-OMe coumarin a 7-OEt coumarin b 7-OMe coumarin c 7-OEt coumarin d 1A2 10. 6 ± 0.4 9.5 ± 2.0 9.4 ± 0.4 6.1 ± 1.4 2E1 7.7 ± 0.5 4.2 ± 1.0 9.6 ± 0.2 6.1 ± 1.1 a A 1 : 1 molar mixture of d 0 and [methyl-d 3 ] 7-OMe coumarin was used as the substrate, and the kinetic isotope effect was measured from the ratio of d 2 ⁄ d 0 formaldehyde product. b A 1 : 1 molar mixture of d 0 and [1-ethyl-d 2 ] 7-OEt coumarin was used as the substrate, and the kinetic isotope effect was measured from the ratio of d 1 ⁄ d 0 acetaldehyde product. c [Methyl-d 2 ] 7-OMe coumarin was used as the substrate, and the kinetic isotope effect was measured from the ratio of d 2 ⁄ d 1 formaldehyde product (multiplied by 2 for the statistical effect). d [1-Ethyl-d 1 ] 7-OEt coumarin was used as the substrate, and the kinetic isotope effect was measured from the ratio of d 1 ⁄ d 0 acetaldehyde product. K H. Kim et al. P450 coumarin dealkylation isotope effects FEBS Journal 273 (2006) 2223–2231 ª 2006 The Authors Journal compilation ª 2006 FEBS 2225 Fig. 2. Steady-state kinetics of 7-OR coumarin O-dealkylation by P450 1A2. (A) 7-OEt coumarin. Steady-state experiments were performed with d 0 (l) and 1,1-d 2 -ethyl (k) substrates. (B) 7-OMe coumarin. Steady-state experiments were performed with d 0 (l)andO-methyl d 3 (k) substrates. The formation of 7-OH coumarin was measured using HPLC. See Table 2 for parameters. Fig. 3. Steady-state kinetics of 7-OR coumarin O-dealkylation by P450 2E1. (A) 7-OEt coumarin. Steady-state experiments were performed with d 0 (s) and 1,1-d 2 -ethyl (d) substrates. (B) 7-OMe coumarin. Steady-state experiments were performed with d 0 (l) and O-methyl d 3 (k) substrates. The formation of 7-OH coumarin was measured using HPLC. See Table 2 for parameters. Table 2. Rates of 7-OEt coumarin O-deethylation and 3-hydroxylation and intermolecular noncompetitive isotope effects. P450 7-OEt coumarin substrate O-Deethylation 3-Hydroxylation k cat (min )1 ) K m (lM) k cat ⁄ K m D V D (V ⁄ K) k cat (min )1 ) K m (lM) k cat ⁄ K m D V D (V ⁄ K) 1A2 d 0 2.0 ± 0.1 6 ± 1 0.34 ± 0.05 8.0 ± 1.0 16 ± 6 14 ± 1 27 ± 6 0.51 ± 0.11 0.92 ± 0.08 0.81 ± 0.25 d 2 0.25 ± 0.02 12 ± 4 0.02 ± 0.01 15 ± 1 24 ± 5 0.64 ± 0.13 2E1 d 0 1.6 ± 0.2 480 ± 90 0.0033 ± 0.0007 2.2 ± 0.9  4 a 0.95 b d 2 0.7 ± 0.3 1300 ± 700 0.00057 ± 0.00004 a Estimated from slopes of v versus S plots (Fig. 3A). b Apparent at [S] ¼ 400 lM. P450 coumarin dealkylation isotope effects K H. Kim et al. 2226 FEBS Journal 273 (2006) 2223–2231 ª 2006 The Authors Journal compilation ª 2006 FEBS when high kinetic isotope effects occurred. This result is in contrast to the case of 7-OMe coumarin with P450 2A6 [19] (but not 7-OEt coumarin [19]). Harada et al. [21] reported trace 6-hydroxylation of 7-OEt cou- marin in rat liver microsomes and a strong switch to this alternate product as a result of deuterium substitution. No other products were detected in this work (see Supplementary material). In other experiments which are not presented, recom- binant human P450 1A1 was used in some experiments, because some work had shown catalytic activity toward 7-OEt coumarin [23]. Rates for 7-OMe coumarin O-demethylation were very low ( 0.02 min )1 ); 7-OEt O-deethylation rates were higher (k cat 0.45 min )1 ) but too low to obtain reliable measurements for the intramolecular and intermolecular competitive experi- ments. For 7-OEt coumarin O-deethylation, the values D V ¼ 3.0 ± 0.2 and D (V ⁄ K) ¼ 2.5 ± 0.9 were obtained. The rate of formation of 3-hydroxy, 7-OEt coumarin was 10-fold slower. Apparent intermolecular noncompetitive kinetic isotope effects in human liver microsomes and a baculovirus-based recominant system with over-expressed NADPH-P450 reductase One possibility to consider is that the rate-limiting nat- ures of reaction steps are perturbed in systems invol- ving purified P450 enzymes that are reconstituted with more NADPH-P450 reductase (EC 1.6.2.4) than is normally associated with the P450s in microsomal membranes [20]. Relatively few studies have considered comparisons because of the issue of multiple P450s cat- alyzing a reaction of interest in microsomes. Miwa et al. [22] reported lack of attenuation of the kinetic isotope effect for 7-OEt coumarin O-deethylation in noncompetitive studies with liver microsomes prepared from 3-methylcholanthrene-treated hamsters, which at that time was considered unusual in that the isotope effects were not well-expressed in other systems. In pre- vious work in this laboratory we analyzed rat P450 1A2 and found similar values for the isotope effects for 7-OMe resorufin O-demethylation, considered to be a relatively selective P450 1A2 substrate, with the purified enzyme and microsomes (assuming that other P450s do not catalyze this reaction at appreciable rates) [18]. The high noncompetitive isotope effects seen in the systems comprised of purified enzymes were also seen in insect cell microsomes from a baculovirus-based expression system in which NADPH-P450 reductase is over-expressed (Table 4). The high values for noncom- petitive intermolecular kinetic isotope effects for the O-dealkylation of both 7-OMe and 7-OEt coumarin are similar to those measured with the reconstituted systems (Tables 2 and 3). High noncompetitive isotope effects were also seen in human liver microsomes (Table 5, Supplementary material). These results are consistent with the earlier conclusions that P450 1A2 is the major enzyme involved in 7-OEt coumarin O-deethylation in human Table 3. Rates of 7-OMe coumarin O-demethylation and 3-hydroxylation and intermolecular noncompetitive isotope effects. P450 7-OMe coumarin substrate O-Demethylation 3-Hydroxylation k cat (min )1 ) K m (lM) k cat ⁄ K m D V D (V ⁄ K) k cat (min )1 ) K m (lM) k cat ⁄ K m D V D (V ⁄ K) 1A2 d 0 4.1 ± 0.3 31 ± 8 0.13 ± 0.04 15 ± 2 8.0 ± 4.0 7.7 ± 0.4 22 ± 4 0.35 ± 0.06 1.1 ± 0.1 0.54 ± 0.14 d 3 0.23 ± 0.02 13 ± 7 0.016 ± 0.006 7.3 ± 0.3 11 ± 2 0.65 ± 0.12 2E1 d 0  2.8  3 a  1.1 d 3 a See Fig. 3(B). Table 4. Rates of 7-OR coumarin O-dealkylation and 3-hydroxylation and intermolecular noncompetitive isotope effects using human P450 1A2 and NADPH-P450 reductase expressed in a baculovirus-based system. Alkoxy coumarin O-Dealkylation 3-Hydroxylation Substrate k cat (min )1 ) K m (lM) k cat ⁄ K m D V D (V ⁄ K) k cat (min )1 ) K m (lM) k cat ⁄ K m D V D (V ⁄ K) 7-OEt d 0 2.0 ± 0.1 8.0 ± 2.0 0.25 ± 0.06 8.3 ± 0.5 6.9 ± 2.4 11 ± 1 11 ± 1 1.0 ± 0.1 0.79 ± 0.09 1.1 ± 0.2 d 2 0.24 ± 0.01 6.7 ± 1.7 0.036 ± 0.009 14 ± 1 15 ± 2 0.90 ± 0.10 7-OMe d 0 4.1 ± 0.1 5.0 ± 0.7 0.82 ± 0.12 10 ± 1 6 ± 2 13 ± 1 33 ± 2 0.40 ± 0.04 1.4 ± 0.20 0.60 ± 0.10 d 3 0.40 ± 0.02 3.0 ± 1.0 0.13 ± 0.04 9.0 ± 1.0 13 ± 2 0.70 ± 0.10 K H. Kim et al. P450 coumarin dealkylation isotope effects FEBS Journal 273 (2006) 2223–2231 ª 2006 The Authors Journal compilation ª 2006 FEBS 2227 liver microsomes at lower 7-OEt coumarin concentra- tions [23]. At very high concentrations of substrate, a lower isotope effect might be expected due to the con- tribution of P450 2E1, an enzyme with a higher K m [23] (Tables 2 and 3). The high noncompetitive isotope effects are interpreted to mean that the step(s) invol- ving the chemistry of C-H bond cleavage are rate- limiting in the reaction even in liver microsomes, as opposed to steps involving generation of the reactive oxygenated iron species. Conclusions The human P450s that have been most implicated in 7-OEt coumarin oxidation were analyzed for kinetic isotope effects in O-dealkylation, as well as with the alternative substrate 7-OMe coumarin. P450 1A2 clearly showed the highest apparent intrinsic kinetic isotope effect, which was not considerably attenuated in various experimental systems, even in liver micro- somes. These results indicate that the rate of C-H bond-breaking is a major factor in determining the rates of the reactions under all conditions. Some shift- ing of the oxidations to the alternate 3-hydroxylation reactions occurs with some of the enzymes (with 7-OMe and 7-OEt coumarin), but not to the extent to utilize all of the electrons that are delivered into the P450 system. P450 2E1 did not show full expression of the isotope effect, and the possibility of rate-limiting steps other than C-H bond breaking is suggested. Earlier work with human P450 2E1-catalyzed oxidations of ethanol and acetaldehyde demonstrated rate-limiting steps fol- lowing product formation and a resulting kinetic iso- tope effect on K m but not k cat [32,33]. This possibility has not been evaluated with 7-OR coumarins and P450 due to the lower rates and the difficulty of estimating k cat and K m (Fig. 3). Another possible complication is the oxidation of the product acetaldehyde by P450 2E1 [33] and the effect on the apparent kinetic constants, which has not been considered in detail here because the primary phenolic products are being measured. To summarize, a major conclusion of the work per- formed with several types of kinetic isotope effects is that P450 1A2 is a major catalyst of the model alkoxy- coumarin reactions in human liver. The actual sub- strate oxidation step is rate-limiting, as opposed to steps involved in the generation of the reactive enzyme-oxygen complex, even in the microsomal sys- tem, and these results should apply to in vivo consider- ations. The conclusions may apply to other P450 reactions, at least to those catalyzed by P450 1A2. Experimental procedures Chemicals 7-OMe and 7-OEt coumarin were purchased from Sigma- Aldrich (Milwaukee, WI, USA) and recrystallized from EtOH-H 2 O mixtures before use. The deuterated substrates were prepared and characterized as described elsewhere [19]. The syntheses and characterization of 3-hydroxy, 7-OMe coumarin and 3-hydroxy, 7-OEt coumarin are described elsewhere [19]. Enzymes Human liver samples were obtained through Tennessee Donor Services, stored at )80 °C, and used to prepare microsomal samples [34]. Human P450s 1A2 [35], 2E1 [36], and 1A1 [37] were expressed in Escherichia coli and purified using modifica- Table 5. Rates of 7-OEt coumarin O-deethylation and intermolecular noncompetitive isotope effects in human liver microsomes. Sample code no. 7-OEt coumarin substrate a O-Deethylation 3-Hydroxylation V b (nmol product ⁄ nmol P450 min )1 ) D V V b (nmol product ⁄ nmol P450 min )1 ) D V 104 d 0 0.048 8.1 0.018 0.95 d 2 0.0059 0.019 109 d 0 0.25 8.1 0.17 1.0 d 2 0.31 0.17 110 d 0 0.14 7.8 0.077 0.70 d 2 0.18 0.11 115 d 0 0.17 7.1 0.12 0.86 d 2 0.024 0.14 132 d 0 0.19 7.3 0.093 0.66 d 2 0.026 0.14 a Used at concentration of 100 lM in all experiments. b Results are expressed as means of duplicate experiments, which differed < 10%. P450 coumarin dealkylation isotope effects K H. Kim et al. 2228 FEBS Journal 273 (2006) 2223–2231 ª 2006 The Authors Journal compilation ª 2006 FEBS tions of the procedures described elsewhere [38]. Insect cell microsomes from P450 1A2 ⁄ baculovirus infections (Super- somes Ò ) were purchased from BD Gentest (Woburn, MA, USA). Rat NADPH-P450 reductase was expressed in E. coli and purified as described [39]. Human b 5 was expressed in E. coli JM109 cells from a plasmid (pSE420 (Amp)) provided by S Asaki (Takeda Pharmaceutical, Osaka, Japan). The protein was purified to electrophoretic homogeneity using modifications of the DEAE-cellulose and other chromatography methods des- cribed elsewhere [19,40]. Enzyme assays Typical steady-state coumarin oxidation reactions included 50 pmol P450, 100 pmol of NADPH-P450 reductase, 50 pmol of b 5 , and 30 lg of di-12 : 0 GPC in 0.50 mL of 50 mm potassium phosphate buffer (pH 7.4) along with a specified amount of the coumarin substrate. In some cases, human liver microsomes (50 pmol P450 in 0.50 mL of 50 mm potassium phosphate buffer, pH 7.4) or insect cell microsomes from a baculovirus-based system contain- ing human P450 1A2 and an excess of NADPH-P450 reductase (BD Gentest Supersomes Ò , 20 pmol P450 in 0.50 mL of 50 mm potassium phosphate buffer, pH 7.4) were used instead of the recombinant (bacterial) P450 sys- tem. An aliquot of an NADPH-generating system was used to start reactions (final concentrations, 10 mm glucose 6- phosphate, 0.5 mm NADP + , and 1 IU yeast glucose 6- phosphate per mL [34]). 7-OMe and 7-OEt coumarin stocks (50 mm) were made in CH 3 CN and diluted into enzyme reactions, with final organic solvent concentrations < 1% (v ⁄ v). Incubations were generally performed for 5–10 min at 37 °C, terminated with 0.10 mL of 17% HClO 4 , and centri- fuged (10 3 g, 10 min). CH 2 Cl 2 (1.0 mL) was added to the supernatant to extract the products followed by centrifuga- tion at 10 3 g (process repeated one more time). The organic layers were combined, and the CH 2 Cl 2 was removed under aN 2 stream. The products, 7-hydroxy coumarin and 3-hydroxy, 7-OR coumarin, were analyzed by HPLC using a Toso ODS-80 TM octadecylsilane (C 18 ) column (4.6 mm 150 mm, 5 lm) with the mobile phase H 2 O:CH 3 CN (55 : 45, v ⁄ v) containing 10 m m HClO 4 , a flow rate of 1.0 mLÆmin )1 , and monitoring at A 330 [19]. Kinetic parame- ters (K m and k cat ) were determined using nonlinear regres- sion analysis with Graph-Pad prism software (Graph-Pad, San Diego, CA, USA). See Supplementary material for typical chromatograms. Intermolecular competitive and intramolecular noncom- petitive kinetic isotope effects were estimated by analysis of the mass spectra of 2,4-dinitrophenylhydrazone derivatives, using the calculation methods previously described [19,41]. The substrate concentration used in these experiments was 50 lm with P450 1A2 and 300 lm with P450 2E1. Mass spectra were recorded using HPLC-MS methods in the Vanderbilt facility with a Thermo-Finnigan TSQ 7000 instrument (Thermo-Finnigan, Sunnyvale, CA, USA) using a Zorbax octadecylsilane (C 18 ) column (6.2 mm · 80 mm, 3 lm) with a mobile phase of H 2 O:CH 3 CN (46 : 54, v ⁄ v) and a flow rate of 2 mLÆmin )1 . The flow was split after the column to result in a flow rate of 1 mLÆ min )1 directed to the mass spectrometer. Deuterium incorporation was deter- mined using negative ion atmospheric pressure chemical ionization MS (source temperature 550 °C, heated capillary temperature 180 °C, heated capillary voltage )20 V, tube lens voltage )40 V, ionization current 5 lA, sheath gas (N 2 ) pressure 70 psi, auxiliary gas (N 2 ) pressure 10 p.s.i). 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Arch Biochem Biophys 350, 324–332. 40 Shimada T, Misono KS & Guengerich FP (1986) Human liver microsomal cytochrome P-450 mepheny- toin 4-hydroxylase, a prototype of genetic polymorph- ism in oxidative drug metabolism. Purification and characterization of two similar forms involved in the reaction. J Biol Chem 261, 909–921. 41 Kolliker S, Oehme M & Dye C (1998) Structure elucida- tion of 2,4-dinitrophenylhydrazone derivatives of carbo- nyl compounds in ambient air by HPLC ⁄ MS and multiple MS ⁄ MS using atmospheric chemical ionization in the negative ion mode. Anal Chem 70, 1979–1985. Supplementary material The following supplementary material is available online: Fig. S1. HPLC traces (A 330 ) for reactions of purified, reconstituted P450 1A2 and 2E1 with 7-OEt coumarin (d 0 and d 2 ). Fig. S2. HPLC traces (A 330 ) for reactions using five human liver microsomal samples with 7-OEt coumarin (d 0 and d 2 ). A blank reaction (no NADPH) is shown for d 2 subsrate. This material is available as part of the online article from http://www.blackwell-synergy.com K H. Kim et al. P450 coumarin dealkylation isotope effects FEBS Journal 273 (2006) 2223–2231 ª 2006 The Authors Journal compilation ª 2006 FEBS 2231 . Kinetic deuterium isotope effects for 7-alkoxycoumarin O-dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes Rate-limiting. a gem secondary kinetic isotope effect has the effect of attenuating the rate of C-H bond- breaking, so in prin- ciple the intrinsic kinetic isotope effect